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Recombinant Human Growth Hormone
Overview Recombinant DNA is DNA that has been constructed in a laboratory and usually contains two or more pieces of DNA fragments that have been ligated together from different species. Similarly, recombinant human growth hormone (hGH), also known as somatropin, is produced by creating a plasmid that contains the gene responsible for hGH production in humans. The plasmid is then expressed in a host bacteria such as ''E. coli. ''The host bacteria acts as a production vehicle by producing the hGH protein from the encoded gene contained within the plasmid. The produced hormone is then extracted from the bacterial culture and can be purified for use in humans. Prior to the production of hGH in bacteria, the hormone was harvested from cadavers. However some patients who recieved cadaver harvested hGH began contracting Creutzfeldt–Jakob disease, often referred to as the human form of mad cow disease. This caused cadaver harvested hGH to be removed from the market and replaced by the bacterial produced version described above.Therefore, a big advantage of recombinant hGH is improved safety for patients. Another advantage of using bacterial machinery, rather than limited cadaver sources, is that production can be increased if supply cannot meet patient demand. . Uses hGH is important in a wide range of bodily functions including metabolism, cell growth, development and immunity. hGH deficiency can occur in humans leading to the need for supplements to be taken in order to correctly balance hGH levels in the body. The hormone can also be used in short-stature children or as a novel method to accelerate wound healing in people with injuries to their bone, cartilage or muscle. Rezaei, Marzieh, and Sayyed H. Zarkesh-Esfahani. "[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3685787/ Optimization of production of recombinant human growth hormone in Escherichia coli.]" ''Journal of research in medical sciences: the official journal of Isfahan University of Medical Sciences'' 17.7 (2012): 681. hGH has a wide range of applications and could be used to treat people in many situations. Until the 1990s, no long term studies had been done to determine what would happen to a patient if he/she was using recombinant hGH for a long period of time (~10 years). A group of scientists from the UK performed this work on a group of 21 adults that were deficient in growth hormone. Gibney, J., et al. "[http://press.endocrine.org/doi/abs/10.1210/jcem.84.8.5916 The effects of 10 years of recombinant human growth hormone (GH) in adult GH-deficient patients]." ''The Journal of Clinical Endocrinology & Metabolism'' 84.8 (1999): 2596-2602. 10 patients received continuous growth hormone replacement, while the other 11 did not. Not surprisingly, they found that adults treated with hGH had an increased lean body and muscle mass, Those patients also had a less atherogenic lipid profile, reduced carotid intima media thickness and an increased psychological well-being. Overall, they found that the patients treated with hGH were doing much better than those who were not being treated, with relatively no bad side effects of long-term use. Production The gene that is responsible for hGH production is ''GHN'' located at chromosome 17q22–24. This gene is cloned together with a vector that is capable of being induced at a high copy number in ''E. coli, ''and a genetic tag that aids in the protein purification process. The plasmid is then transformed into the competent host cell by either applying an electrical or heat shock to the cells. This process creates disruptions in the cell membrane by which the recombinant DNA can be acquired into the cell. At this point, the cells that do not contain a plasmid need to be separated from those that do contain a plasmid. Usually, the plasmids that are being transformed contain an antibiotic resistance gene that is expressed in the cells that contain the plasmid. Cells not containing the plasmid will die when stressed with the specific antibiotic contained on the plasmid. After selecting for cell colonies that contain the hGH producing plasmid, cells are grown overnight at 37C to an OD600 = 0.7. From this culture a new culture is inoculated and IPTG is added to a final concentration of 1mM. IPTG is responsible for inducing the promoter that controls the ''GHN gene ''on the plasmid. This new culture is grown at 32C for five hours and are then centrifuged to concentrate the cells into a pellet for hGH harvesting. References